Anthrax, the disease caused by the spore-forming Bacillus anthracis (B. anthracis), continues to be a worldwide problem among domesticated and wild herbivores in Asia and Africa and poses a worldwide threat when being used as a biological weapon for biological warfare or bioterrorism. Human infections occur after contact with infected animals or contaminated animal products. Outbreaks or epidemics are a constant threat for endemic regions because spores can persist in the soil for long periods of time. Importation controls on certain animal products are necessary to prevent the establishment of anthrax where the disease is not endemic. Human anthrax is usually classified by the portal of entry into the host. Cutaneous anthrax, which accounts for the vast majority of human anthrax cases, is a localized infection with generally mild systemic symptoms and characterized by a painless papule that is surrounded by edema which can be quite extensive. The papule ulcerates by day 5 or 6 and develops into the characteristic black eschar of cutaneous anthrax. Inhalation anthrax, which occurs after inhaling airborne spores, gastrointestinal anthrax, resulting from ingestion of contaminated food, and, in some instances, untreated cutaneous anthrax are characterized by dissemination of the bacteria from the initial site of infection with development of a massive septicemia and toxemia. In inhalation anthrax, phagocytic cells transport the spores from the lung alveoli to the regional lymph nodes, where the spores germinate and bacteria multiply. The bacilli then spread into the bloodstream, where they are temporarily removed by the reticuloendothelial system. Prior to death, which occurs 2 to 5 days after infection, there is a sudden onset of acute symptoms characterized by hypotension, edema, and fatal shock due to an extensive septicemia and toxemia. Therapeutic intervention, in general, must be initiated early, as septicemic infections are nearly always fatal.
The invention relates to the modulation of gene expression in a cell, also called gene control, in relation to the treatment of a variety of diseases such as anthrax. As said, anthrax is a disease of animals and humans that poses a significant threat as an agent of biological warfare and terrorism. Inhalational anthrax, in which spores of B. anthracis are inhaled, is almost always fatal, as diagnosis is rarely possible before the disease has progressed to a point where antibiotic treatment is ineffective. The major virulence factors of B. anthracis are a poly-D-glutamic acid capsule and anthrax toxin. Anthrax toxin consists of three distinct proteins that act in concert: two enzymes, lethal factor (LF) and edema factor (EF; an adenylate cyclase); and protective antigen (PA). The PA is a four-domain protein that binds a host cell-surface receptor by its carboxy-terminal domain; cleavage of its N-terminal domain by a furin-like protease allows PA to form heptamers that bind the toxic enzymes with high affinity through homologous N-terminal domains. The complex is endocytosed; acidification of the endosome leads to membrane insertion of the PA heptamer by forming a 14-stranded beta-barrel, followed by translocation of the toxic enzymes into the cytosol by an unknown mechanism. The binary combination of PA and LF is sufficient to induce rapid death in animals when given intravenously, and certain metalloprotease inhibitors block the effects of the toxin in vitro. Thus, LF is a potential target for therapeutic agents that would inhibit its catalytic activity or block its association with PA. LF is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the PA; domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of mitogen-activated protein kinase kinase-2 (MAPKK-2) before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family and contains the catalytic center; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion into an enzyme with high and unusual specificity.
The MAPKK family of proteins is the only known cellular substrates of LF. Cleavage by LF near to its N-terminal removes the docking sequence for the downstream cognate MAP kinase. The effect of lethal toxin on tumor cells, for example, is to inhibit tumor growth and angiogenesis, most probably by inhibiting the MAPKK-1 and MAPKK-2 pathways. However, the primary cell type affected in anthrax pathogenesis is the macrophage. LF has been shown to cleave short N-terminal fragments from mitogen or extracellular signal-regulated MAPKK-1, MAPKK-2, MAPKK-3, and MAPKK-6, the upstream activators of extracellular signal-regulated kinase 1 (ERK1), ERK2, and p38. Recent data show that this results in inhibiting release, but not production, of the pro-inflammatory mediators, NO and tumor necrosis factor-alpha (TNF-alpha). In addition, high levels of lethal toxin lead to lysis of macrophages within a few hours by an unknown mechanism. Recent data suggests that this happens due to inhibition of growth-factor pathways leading to macrophage death. These observations suggest that at an early stage in infection, lethal toxin may reduce (or delay) the immune response, whereas at a late stage in infection, high titers of the bacterium in the bloodstream trigger macrophage lysis and the sudden release of high levels of NO and TNF-alpha. This may explain the symptoms before death which are characterized by the hyperstimulation of host macrophage inflammatory pathways, leading to dramatic hypotension and shock. These symptoms resemble those of LPS-induced septic shock, whereby it is of note that LPS-nonresponder mice such as C3H/HeJ are also quite resistant against anthrax toxin.
The recognition sites for LF require the presence of the proline (P) residue followed by a hydrophobic residue or a glycine (G) residue, between which LF cleaves. The recognition sites further require an uncharged amino acid following the hydrophobic residue and at least one positively charged amino acid (and no negatively charged amino acid, such as Asp and Glu) within the 5 amino acids to the N-terminal side of the proline residue. Other residues in the sequence provide appropriate spacing between the critical residues or between the donor and acceptor, and thus their composition is not critical and can include any natural or unnatural amino acid.
Gene control is generally thought to occur at four levels: 1) transcription (either initiation or termination), 2) processing of primary transcripts, 3) stabilization or destabilization of mRNAs, and 4) mRNA translation. The primary function of gene control in cells is to adjust the enzymatic machinery of the cell to its nutritional, chemical and physical environment.
It is generally thought that gene expression is regulated at both the level of transcription and translation. Modulation or regulation of gene expression requires factors called transcriptional factors. The term “gene control or regulation” also refers to the formation and use of mRNA. Although control can be exerted at a number of different molecular steps, differential transcription probably most frequently underlies the differential rate of protein synthesis in prokaryotes as well as eukaryotes. It is generally thought that activator proteins (also called transcription factors or transcriptional activators) bind to DNA and recruit the transcriptional machinery in a cell to a promotor, thereby stimulating gene expression. Further, differential processing of RNA transcripts in the cell nucleus, differential stabilization of mRNA in the cytoplasm, and differential translation of mRNA into protein are also important in eukaryotic gene control. These steps define the regulatory decisions in a transcriptional circuit and misregulation at any stage can result in a variety of diseases.
Where in unicellular organisms, be it of prokaryotic or eukaryotic origin, a cell's response to its environment is influenced by many stimuli from the outside world, reflecting the often widely variable environment of the single cell, most cells in multicellular organisms experience a fairly constant environment. Perhaps for this reason, genes that are devoted to responses to environmental changes constitute a much smaller fraction of the total number of genes in multicellular organisms than in single-cell organisms.
As said above, cells react to environmental changes, which they perceive through extracellular signals. These signals can be either physical (e.g., light, temperature, pressure and electricity) or chemical (e.g., food, hormones and neurotransmitters). Cells can both sense and produce signals. This makes it possible that they communicate with each other. In order to achieve this, there are complex signal-sensing and -producing mechanisms in uni- and multi-cellular organisms.
Two groups of chemical signals can be distinguished: membrane-permeable and membrane-impermeable signals. The membrane-permeable signal molecules comprise the large family of steroid hormones, such as estrogens, progesterone and androgens. Steroids pass the plasma membrane and bind to specific receptors, which are localized in the cytoplasm or nucleus of the cell. After binding of the hormone, the receptor undergoes a conformational change. The receptor is then able to bind to DNA itself or to proteins which can in turn interact with DNA. In general, steroid hormones can directly regulate gene expression by means of this process. The membrane-impermeable signal molecules include acetylcholine, growth factors, extracellular matrix components, thrombin, lysophosphatidic acid, the yeast mating factors and, for the social amoeba Dictyostellium discoideum, folic acid and cyclic AMP. They are recognized by receptors, which are localized on the plasma membrane of the cell. The receptors are specific for one particular signal molecule or a family of closely related signal molecules. Upon binding of their ligands, these receptors transduce the signals by several mechanisms.
The most characteristic and exacting requirement of gene control on multicellular organisms is the execution of precise developmental decisions so that the right gene is activated in the right cell at the right time. These developmental decisions include not only those related to the development of an organism per se, as, for example, can be seen during embryogenesis and organogenesis, or in response to disease but also relate to the differentiation or proliferation or apoptosis of those cells that merely carry out their genetic program essentially without leaving progeny behind.
Such cells, such as skin cells, precursors of red blood cells, lens cells of the eye, and antibody-producing cells, are also often regulated by patterns of gene control that serve the need of the whole organism and not the survival of an individual cell.
It is generally reasoned that there are at least three components of gene control: molecular signals, levels and mechanisms. First, it is reasoned that specific signaling molecules exist to which a specific gene can respond. Second, control is exerted on one or more levels (i.e., the step or steps) in the chain of events leading from the transcription of DNA to the use of mRNA in protein synthesis. Third, at each of those levels, specific molecular mechanisms are employed to finally exert the control over the gene to be expressed.
Many genes are regulated not by a signaling molecule that enters the cells but by molecules that bind to specific receptors on the surface of cells. Interaction between cell-surface receptors and their ligands can be followed by a cascade of intracellular events including variations in the intracellular levels of so-called second messengers (diacylglycerol, Ca2+, cyclic nucleotides). The second messengers in turn lead to changes in protein phosphorylation through the action of cyclic AMP, cyclic GMP, calcium-activated protein kinases, or protein kinase C, which is activated by diaglycerol.
Many of the responses to binding of ligands to cell-surface receptors are cytoplasmatic and do not involve immediate gene activation in the nucleus. Some receptor-ligand interactions, however, are known to cause prompt nuclear transcriptional activation of a specific and limited set of genes. For example, one proto-oncogene, c-fos, is known to be activated in some cell types by elevation of almost every one of the known second messengers and also by at least two growth factors, platelet-derived growth factor and epidermal growth factor. However, progress has been slow in determining exactly how such activation is achieved. In a few cases, the transcriptional proteins that respond to cell-surface signals have been characterized.
One of the clearest examples of activation of a pre-existing inactive transcription factor following a cell-surface interaction is the nuclear factor (NF)-κB, which was originally detected because it stimulates the transcription of genes encoding immunoglobulins of the kappa (κ) class in B-lymphocytes. The binding site for NK-κB in the kappa gene is well defined (see, for example, P. A. Baeuerle and D. Baltimore, 1988, Science 242:540), providing an assay for the presence of the active factor. This factor exists in the cytoplasm of lymphocytes complexed with an inhibitor. Treatment of the isolated complex in vitro with mild denaturing conditions dissociates the complex, thus freeing NK-κB to bind to its DNA site. Release of active NF-κB in cells is now known to occur after a variety of stimuli including treating cells with bacterial lipopolysaccharide (LPS) and extracellular polypeptides as well as chemical molecules (e.g., phobol esters) that stimulate intracellular phosphokinases. Thus, a phosphorylation event triggered by many possible stimuli may account for NF-KB conversion to the active state. The active factor is then translocated to the cell nucleus to stimulate transcription only of genes with a binding site for active NF-κB.
The inflammatory response involves the sequential release of mediators and the recruitment of circulating leukocytes, which become activated at the inflammatory site and release further mediators (Nat. Med. 7:1294;2001). This response is self-limiting and resolves through the release of endogenous anti-inflammatory mediators and the clearance of inflammatory cells. The persistent accumulation and activation of leukocytes is a hallmark of chronic inflammation. Current approaches to the treatment of inflammation rely on the inhibition of pro-inflammatory mediator production and of mechanisms that initiate the inflammatory response. However, the mechanisms by which the inflammatory response resolves might provide new targets in the treatment of chronic inflammation. Studies in different experimental models of resolving inflammation have identified several putative mechanisms and mediators of inflammatory resolution. We have shown that cyclopentenone prostaglandins (cyPGs) may be endogenous anti-inflammatory mediators and promote the resolution of inflammation in vivo. Others have shown a temporal shift to the production of anti-inflammatory lipoxins during the resolution of inflammation. In recent years, apoptosis has been identified as an important mechanism for the resolution of inflammation in vivo. It has been postulated that defects in leukocyte apoptosis are important in the pathogenesis of inflammatory disease. In addition, the selective induction of apoptosis in leukocytes may offer a new therapeutic approach to inflammatory disease.
Considering that NF-KB is thought by many to be a primary effector of disease (A. S. Baldwin, J. Clin. Invest., 2001, 107:3–6), numerous efforts are underway to develop safe inhibitors of NF-κB to be used in treatment of both chronic and acute disease situations. Specific inhibitors of NF-κB should reduce side effects associated with drugs such as NSAIDS and glucocorticoids and would offer significant potential for the treatment of a variety of human and animal diseases. Specific diseases or syndromes where patients would benefit from NF-κB inhibition vary widely, and range from rheumatoid arthritis, atherosclerosis, multiple sclerosis, chronic inflammatory demyelinating polyradiculoneuritis, asthma, inflammatory bowel disease, to Helicobacter pylori-associated gastritis and other inflammatory responses, and a variety of drugs that have effects on NF-κB activity, such as corticosteroids, sulfasalazine, 5-aminosalicylic acid, aspirin, tepoxalin, leflunomide, curcumin, antioxidants and proteasome inhibitors. These drugs are considered to be non-specific and often only applicable in high concentrations that may end up being toxic for the individual treated.
Inactive cytoplasmatic forms of transcription factors can thus be activated by removal of an inhibitor, as is the case with NF-kapppaB, or, alternatively, by association of two (or more) proteins, neither of which is active by itself as in the case of interferon-alpha-stimulated factor (D. E. Levy et al., 1989, Genes and Development 3:1362). After interferon-alpha attaches to its cell-surface receptor, one of the proteins is changed within a minute or less, and the two can combine. The active (combined) factor is then translocated to the cell nucleus to stimulate transcription only of genes with a binding site for the protein. Considering that interferon-alpha is a mediator of responses of the body directed at pathogens and self-antigens, modulating regulation of genes that are under influence of the interferon-alpha-stimulated factor would contribute to the treatment of a variety of human and animal diseases.
Other typical examples of signaling molecules that affect gene expression via cell-surface receptor interaction are polypeptide hormones such as insulin, glucagon, various growth factors such as EGF, VEGF, and so on.
The steroid hormones and their receptors represent one of the best understood cases that affect transcription. Because steroid hormones are soluble in lipid membranes, they can diffuse into cells. They affect transcription by binding to specific intracellular receptors that are site-specific DNA-binding molecules. Other examples of signaling molecules that enter the cell and act intra-cellularly are thyroid hormone (T3), vitamin D and retinoic acid, other small lipid-soluble signaling molecules that enter cells and modulate gene expression. The characteristic DNA-binding sites for the receptors for these signaling molecules are also known as response elements.
Another example of a small molecule that is involved in regulation of gene expression is ethylene, a gas that, for example, induces the expression of genes involved in fruit ripening. Also, small plant hormones, known as auxins and cytokinins, regulate plant growth and differentiation directly by regulating gene expression.
Given the critical role of regulatory factors in gene regulation, the development of artificial or synthetic counterparts that could be used in methods to rectify errors in gene expression has been a long-standing goal at the interface of chemistry and biology.